Characterization and identification of wheat (Triticum aestivum L.) rhizospheric bacteria by using 16s rRNA gene sequencing

J. Bio. Env. Sci. 2016
Journal of Biodiversity and Environmental Sciences (JBES)
ISSN: 2220-6663 (Print) 2222-3045 (Online)
Vol. 9, No. 6, p. 31-38, 2016
http://www.innspub.net
RESEARCH PAPER
OPEN ACCESS
Characterization and identification of wheat
(Triticum aestivum L.) rhizospheric bacteria by using
16s rRNA gene sequencing
Rizwan Ali Sheirdil *1,2 , Rifat Hayat 1 , Xiao-Xia Zhang 2 , Nadeem Akhtar Abbasi 3 ,
Safdar Ali 1 , Muhammad Azeem 1
1
Department of Soil Science, Soil and Water Conservation, PMAS Arid Agriculture University,
Rawalpindi, Pakistan
2
ACCC(Agricultural Culture Collection of China), Institute of Agricultural Resources and Regional Planning,
Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
3
Department of Horticulture, PMAS Arid Agriculture University, Rawalpindi, Pakistan
Article published on December 30, 2016
Key words: Chemotaxonomic characterization, PGPR, Wheat, Yield, Yield attributes
Abstract
Bacterial strains were isolated from wheat rhizospheric soil with the aim to explore potential strains for the
biofertilizer technology suitable to the local climate and conditions. All the isolates were characterized for their
plant growth promoting (PGP) activities such as IAA production, P-solublization, nifH gene presence, catalase
and oxidase production. The promising and potential strains based on their PGP activities were tested in two
experiments of wheat. In the first growth chamber experiment, ten strains were inoculated with the seeds and
sown in small pots filled with the soil. All the strains showed positive results in yield attributes of the plant like
shoot length, root length, fresh and dry weight. Further, six strains which performed best in the growth chamber
experiment were tested in a pot experiment under controlled conditions and the results showed that each strain
significantly increased like shoot length (7.6-25%), root length (19.7-34.1%), number of tillers (33.3-100%), fresh
weight (17.8-31.3%), dry weight (19.3-48.6%), spike length (4.3-28.2%) and grain yield (7.6-41.5%) of wheat
plants over control. The strains used in the pot experiment were identified by standard procedure of 16s rRNA
gene sequencing which showed that two of strains were from genus Bacillus, two from Pseudomonas, one from
Acinetobacter and one from Jeotgalicoccus.
*Corresponding Author: Rizwan Ali Sheirdil rizwan_sheirdil52@yahoo.com
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J. Bio. Env. Sci. 2016
Introduction
Wheat is the main staple food for most of the
population of Pakistan and largest grain source of the
country. It adds 13.1 percent to the value added in
agriculture and 2.7 percent to GDP. The estimated
yield obtained in 2010 was 24.2 million tons
(Economic Survey of Pakistan 2010-11). Average
wheat yield in rainfed area is 1.5 tons per hectare
which is dependent on the amount of rainfall. This is
below the potential yield and the major reasons for
low productivity in rainfed areas are low soil fertility,
shortage of irrigation water and inefficient fertilizer
use. Soils of Pot war are low in organic matter
contents which affect soil fertility and soil structure
badly (PARC). Therefore for sustainable agriculture,
the use of plant growth promoting rhizobacteria
(PGPR) has increased throughout the world.
Inoculation of PGPR has reported the increase in the
yield of many agronomically important field crops
(Biswas et al., 2000a,b; Asghar et al., 2002 and
Khalid et al., 2003).
Plant growth promoting rhizobacteria are a group of
bacteria living free in the soil or in association with
plants and enhance plant growth and yield by
different mechanisms of action. They may produce
different hormones which stimulate plant growth,
solubilize nutrients including phosphorous
solubilization and iron chelation, fix atmospheric
nitrogen, act as bio-control agents and improve soil
structure (Hayat et al., 2010). PGPRs are often
termed as “biofertilizers” as they increase the
availability and uptake of nutrients for the plants
(Vessey, 2003). Biofertilizers are an important
component of integrated plant nutrient management
as they elevate the circulation of plant nutrients and
lessen the need of chemical fertilizers (Rodriguez and
Fraga, 1999).
These biofertilizers include bacterial strains from
genera such as Pseudomonas, Azospirillium,
Azotobacter, Bacillus, Burkholderia, Enterobacter,
Rhizobium, Erwiniaand Flavobacterium,
Acinetobacter and Jeotgalicoccus (Rodriguez and
Fraga, 1999).
Several studies clearly demonstrated the positive and
beneficial effects of PGPR on growth and yield of
different crops. Increases in agronomic yield due to
PGPR inoculation have been correlated to production
of growth-stimulating phytohormones as well as
phosphate solubilisation (Kohler et al., 2006). It has
been reported that wheat yield was enhanced up to
30% with Azotobacter inoculation and up to 43% with
Bacillus inoculation (Kloepper et al., 1991). Similar
results were obtained when barley seed was
inoculated with different PGPRs (Canbolat et al.,
2006). Root weight was increased by 8.9–16.7% and
shoot weight by 28.6–34.7% over the control.
According to Wu et al. (2005) microbial inoculum
Bacillus megaterium and Bacillus mucilaginous
increased the plant growth as well as improved
nutritional assimilation of plant (total N, P, and K).
Egamberdiyeva (2007) inoculated maize with
bacterial strains Pseudomonas alcaligenes, Bacillus
polymyxa, and Mycobacterium phlei and reported a
significant increase in root dry weight (19–52%) and
total dry matter of maize was increased up to 38%.
There was 15 % increase in wheat yield due to
inoculation of PGPR as reported by Chen et al.
(1996). Keeping in view the beneficial effects of
PGPR, a lab study was conducted in to isolate the
bacterial strains from wheat rhizosphere, identified
them and evaluated their efficiency as PGPR on
growth of wheat in growth pouches under controlled
conditions in growth chamber.
Material and methods
Isolation, screening and phenotypic characterization
of soil bacteria
Bacterial strains were isolated from wheat
rhizospheric soil obtained from Abdullah Shah Ghazi
Bukhari, Attock (33 o 14’26.38” N and 72 o 23’10.29”
E)which has sandy loam soil. Isolation of the stains
was carried out by dilution plate technique using
phosphate buffer saline as a saline solution. Tryptic
Soya Agar (TSA; Difco) medium was used and the
plates used for isolation were incubated at 28ᵒC for
48-72 hours depending upon the growth of the
bacterial colonies.
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J. Bio. Env. Sci. 2016
Then single bacterial colonies were picked and
streaked on TSA medium plates with the aim to
achieve single colonies. When single colonies were
achieved, these were preserved in 35% glycerol (w/v)
at -80 o C and further characterization was carried out.
The morphological characteristics of the strains were
observed and noted using light microscope (Olympus
BX 60).
Plant growth promoting assay
Plant growth promotion activities like IAA production,
phosphorus solubalization and presence of nifH gene
were evaluated following different standard
procedures. For IAA production, bacterial cultures
were grown in Tryptic Soya Broth (TSB) at 28±2 °C for
24-36 hours. Then 100 uL of this bacterial suspension
was inoculated in 5 ml of autoclaved Luria Broth
medium (LB medium) with and without L-tryptophan.
L-tryptophan was added 500 µg ml -1 in each test tube.
These test tubes with the LB medium and bacterial
suspension were allowed to grow at 28±2 °C in an
incubator shaker for 48 hours. After 48 hours, these
cultures were centrifuged at 10000 rpm for 10 minutes.
2 ml supernatant was then mixed with two drops of
Orthophosphoric acid and 4ml of the Salkowski
reagents (50ml, 35% of Perchloric acid, 1ml 0.5M FeCl3
solution) and the optical density was determined at
530 nanometer using spectrophotometer.
Development of pink color was an indicator of IAA
production. IAA production by strains was measured
by standard curve graph where standards range was
upto 10µg ml-1 (Brick et al., 1991).
P-solublization was determined qualitatively by
measuring the halo zone formation by bacteria on
Pikovskaya (PKV) agar medium (Pikovskaya, 1948;
Gaur, 1990) and the P-solublizer strains were further
evaluated quantitatively. Quantitative P-
solubalization was measured by inoculation of
bacterial broth culture in a 250 ml flask containing
100 ml of PKV medium and 5 g L-1 of insoluble tricalcium
phosphate. The pH of the media was adjusted
at 7.0 and was autoclaved at 121 o C for 15 minutes.
After the inoculation of bacterial culture, the flasks
were placed in the incubator shaker for 8 days at
30 o C. The pH of the media was measured followed by
centrifugation at 8500 rpm for 25 min. The
supernatant was measured for available phosphorus
by the protocol given by Watanabe & Olsen (1965).
The optical density of the supernatant was
determined at 700 nm using spectrophotometer and
the values were determined by standard curve graph
and standards range was up to 1 µg ml -1 .Nitrogen
fixing characteristic of the bacterial strains was
checked by amplification of nifH gene by PCR
amplifications. Universal forward and reverse
primers PolF b (TGC GAY CCS AAR GCBGACTC) and
PolR b (ATSGCCATCATYTCRCCG GA) were used,
respectively.
Identification of bacterial strains using 16SrRNA
gene sequencing
Standard method of 16S rRNA gene sequencing was
used to identify the strains. Singe colony of each
strain was picked and DNA template was prepared
followed by 16S rRNA amplification with help of PCR.
Universal primers 9F (5́-
GAGTTTGATCCTGGCTCAG-3́) and 1510R (5́-
GGCTACCTTGTTACGA-3́) were used for PCR
amplification. For the 16S rRNA gene amplification,
the reaction mixture (25 µL) was prepared by initial
denaturation for 2 minutes at 94°C. Further,30 cycles
of denaturation at 94 °C for one minute was carried
out followed by primer annealing at 52°Cfor half a
minute. After annealing, the primer extension was
carried out at 72°C for 2 min and at last final
extension at 72°C for 10 min in a thermo cycler.
Amplified PCR products of 16S ribosomal gene were
separated on 1% agarose gel in 0.5X TE (Tris-EDTA)
buffer containing 2 µL ethidiumbromide (20 mg/mL).
λ Hind-III ladder was used as a size marker. The gel
was viewed under UV light and photographed using
gel documentation system. Amplified PCR products
of full-length 16S rRNA genes were purified using
PCR purification kit (QIAGEN) according to the
standard protocol recommended by the
manufacturer.
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J. Bio. Env. Sci. 2016
The purified PCR product samples were sequenced
using DNA sequencing service of MACROGEN, Korea
(www.dna.macrogen.com/eng) using universal 16Sr
RNA gene sequencing primers. The sequence results
were blast through NCBI/Eztaxon (Chun et al., 2007)
and sequence of all the related species were retrieved
to get the exact nomenclature of the isolates.
Phylogenetic analyses were performed using
bioinformatics software MEGA-5 (Tamura et al.,
2007). Other software used for sequence alignment
and comparisons were CLUSTAL X and Bio-Edit.
DNA accession numbers of each strain were obtained
from DNA Data Bank of Japan (DDBJ).
Wheat inoculation
Among all the isolates, potential strains on the basis
of PGP tests were selected for evaluation of their
effect on wheat crop. Initially ten strains were
selected and were inoculated in the growth chamber
experiment (controlled conditions) which was
conducted in small pots having sterilized soil. Uninoculated
pots were treated as control. There were 11
treatments including the control with four
replications with CRD as the experimental design as
the experimental units were homogeneous. Plant
parameters like shoot length, root length, fresh and
dry weight (oven dried at 65 o C for 24 hours) were
measured after a month of germination and most
promising six strains were further evaluated in pot
experiment using CRD design. There were seven
treatments in the pot experiments including the uninoculated
treated as control with four replications.
Plant yield and yield attributes like shoot length, root
lengths, number of tillers, fresh weight (gm/plant),
dry weight (gm/plant), spike length were measured at
the harvest maturity of the crop. The obtained data
was analyzed statistically by ANOVA and the means
were compared using LSD test with significance level
of ≤0.05 (Steel et al., 1997).
Results and discussion
Among all the strains isolated from wheat
rhizosphere, the potential strains which produced
maximum indole acetic acid and solubilized
phosphorus were checked for growth enhancing
parameters on wheat crop in two experiments.
Table 1. Impact of Inoculum on plant growth (Growth chamber experiment).
S. No. Strain ID Treatment Shoot length (cm) Root length (cm) Fresh weight (gm) Dry weight (gm)
1 Control T1 15.9±2.23 5.13±0.93 1.47±0.46 0.76±0.15
2 RW-45 T2 24.8±3.15 7.54±1.03 2.17±1.21 1.24±0.45
3 RW-14 T3 22.1±2.96 7.69±1.13 3.99±1.94 2.09±1.09
4 RW-46 T4 24.4±3.49 10.25±2.21 3.75±1.90 2.14±1.11
5 RW-43 T5 24.7±3.41 10.81±2.06 2.99±1.14 1.68±1.34
6 RW-17 T6 22.3±3.06 8.96±1.04 2.64±1.19 1.37±1.22
7 RW-34 T7 23.2±3.45 13.37±3.17 4.01±3.54 1.98±2.06
8 RW-13 T8 21.9±3.55 10.34±2.03 2.40±1.09 1.16±1.06
9 RW-35 T9 25.7±3.97 11.25±1.45 2.53±1.57 1.35±1.17
10 RW-33 T10 22.9±3.19 9.68±1.24 3.23±2.06 1.81±1.31
11 RW-42 T11 27.7±4.09 12.91±2.19 4.26±2.19 2.29±1.09
The first experiment was carried out in growth
chamber in controlled conditions for one month and
ten strains were inoculated with seed and sown. All
the parameters taken showed the positive results with
increase in shoot length, root length, fresh and dry
weight of plants (Table 1). Significant increase in
shoot length was observed in all the treatments over
control.
Maximum increase was observed in T11, which gave
74% increase in shoot length followed by 61%, 56%,
55%, 53% by T9, T2, T5 and T4, respectively. Khalid
et al., 2003 observed 70.5% increase in shoot length
of wheat by application of a strain isolated from
wheat rhizosphere. This increase in the shoot length
can be due to release of metabolites by PGPRs (Van
Loon, 2007) and mineralization of nutrients which
are easily available for plants.
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J. Bio. Env. Sci. 2016
An increase of 159% in root length was observed by T7
followed by 151%, 119%, 110% and 100% by T11, T9,
T5 and T8, respectively when compared to control
(Fig. 1). Shaharooma et al., 2008 reported that there
is significant increase in root length due to
application of PGPRs; they also state that phytohormones
production by PGPRs can be major cause
of increase in root length of plants. Increase in fresh
and dry weight of the plants was observed in all the
treatments over control.
Table 2. Impact of Inoculum on plant growth (Pot experiment).
S. No. Treatment Strain ID Shoot length Root length No. of Fresh weight Dry weight Spike length Grain yield (kg/ha)
(cm) (cm) Tillers (gm/plant) (gm/plant) (cm)
1 T1 Control 71.26±7.16 16.56±2.35 3 17.72±2.54 6.64±1.23 6.70±0.99 2900.43±203.15
2 T2 RW-46 76.67±8.16 19.82±3.15 4 21.98±4.19 8.63±2.65 7.55±1.27 3119.82±249.25
3 T3 RW-43 82.49±9.68 20.97±4.15 4 20.87±2.15 7.92±1.19 6.99±1.09 3158.98±293.65
4 T4 RW-45 76.09±8.49 21.87±4.58 5 22.52±2.19 8.32±2.19 7.19±2.14 3612.36±353.98
5 T5 RW-35 84.82±8.98 21.56±4.19 4 22.55±2.48 8.36±2.22 7.51±2.06 3906.76±393.65
6 T6 RW-42 89.05±9.58 22.21±5.16 6 23.27±3.78 8.66±2.14 8.59±2.58 4103.22±416.35
7 T7 RW-34 85.46±8.47 21.57±4.78 5 22.48±2.98 9.87±3.09 7.85±2.77 3705.54±347.85
The maximum increase in fresh weight was 189% by
T11 followed by 172%, 171%, 155% by T7, T3 and T4.
Pras hant D et al., 2009 reported an increase in dry
weight of wheat plants by application of PGPRs.
The second experiment was carried out in plastic pots
in glass house (Table 2). Six strains which showed
maximum growth in the first experiment were selected
and inoculated with seed. The data taken at harvest
showed positive results in every parameter by all the
PGPRs application over control. The results correlate
significantly with findings of Saber et al., 2012.Shoot
length was measured at the time of harvest.
The results showed that T6 had an increase of 25% in
shoot length over control followed by T7, T5 and T3
which showed an increase of 20%, 19% and 16%
respectively. There was significant increase in shoot
length of wheat plants sue to application of PGPRs
when applied with combination of chemical fertilizers
and composts (Akhtar et al., 2009). Along with the
increase in shoot length, there was increase in root
length too. An increase of 34% was observed in root
length by inoculation of T6 in the pot experiment
followed by 30% by T5 and T7. The root length was
increased significantly when different rhizobacteria
were inoculated on wheat plants in the pot
experiment (Khalid et al., 2004).
The increase in the root length can be due to the
interaction of PGPRs with the roots and increased cell
division in the root tips (Levanony and Bashan,
1989). Similar results have been observed in maize
(Kapulnik and Okon, 1983) and tomato (Hadas and
Okon, 1987).
The PGPRs also had positive effect on the number of
tillers as reported by Afzal et al., 2005. A 100%
increase in number of tillers plant -1 was showed by T6
followed by 66% increase by T4 and T7 and 33% by
T2, T3 and T5. Sisie et al., 2011 reported an increase
of 25% in number of tillers in wheat by the
application of PGPRs. The production of IAA by the
rhizobacteria has been discussed as the cause of
increase in tillers of the plant but still this factor
cannot be the only one reason. The other reason can
be the general growth enhancement of the plants by
PGPRs Assuero and Jorge, 2010.
Fresh and dry weight plant -1 is a major parameter to
determine the growth of the plants. Inoculation of
PGPRs significantly enhanced the fresh and dry
weight of the plants. Maximum increase in fresh
weight plant -1 was observed by T6 which was 31%
more than control. An increase of 27% in fresh weight
was observed by T4 and T5 followed by 26% by T7
and 24% by T1.
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J. Bio. Env. Sci. 2016
Dry weight of the plants also showed similar results to
the fresh weight with only one variation as maximum
increase was showed by T7 followed by T6 and T2.
Increase in dry weight of the wheat plants was stated
by Egamberdieva, 2010, Narula et al., 2006. Spike
length was also measured and was found correlated to
other parameter findings. T6 exhibited longest spikes
as compared to all other treatments.
Fig. 1. Impact of inoculum application on Plant height (cm).
There was 28% increase in the length of the spikes in
the T6 when compared with the control. T7 showed
17% increase, followed by 12% by T2 and T5. Afzal
and Asghar, 2008 and Agamy et al., 2012 reported
increase in length of the spike by application of
PGPRs. Grain yield of the wheat plants was positively
influenced by application of PGPR strains.
All the strains showed significant increase over
control. Maximum increase in grain yield was
observed by T6 which was 41% more than the
control. Other treatments had 34%, 27% and 24%
increase n grain yield by T5, T7 and T4 respectively.
Increase in grain yield of wheat plants due to
application of PGPRs has been stated by Faramarzi
et al., 2012, Khalid et al., 2004 and Ozturk et al.,
2003. As the number of tillers plant -1 were
increased by application of PGPRs so it can have
effect on grain yield.
Acknowledgement
The research article is a part of Ph.D. research work,
and the first author profoundly acknowledges the full
cooperation of supervisory committee and their
valuable suggestions during the whole experimental
durations. Financial support from the Higher
Education Commission Pakistan (PhD. Indigenous
Scholarship) is also greatly acknowledged.
References
Abbaspoor A, Zabihi HR, Movafegh S, Akbari
MH. 2009. The efficiency of Plant Growth Promoting
Rhizobacteria (PGPR) on yield and yield components
of two varieties of wheat in salinity condition.
American-Eurasian Journal of Sustainable
Agriculture 3, 824-828.
Afzal A, Ashraf M, Asad SA, Farooq M. 2005.
Effect of phosphate solubilizing microorganisms on
phosphorus uptake, yield and yield traits of wheat
(Triticum aestivum L.) in rainfed area. International
Journal of Agriculture & Biology 7, 207-209.
36 | Sheirdil et al.

J. Bio. Env. Sci. 2016
Afzal A, Bano A. 2008. Rhizobium and phosphate
solubilizing bacteria improve the yield and phosphorus
uptake in wheat (Triticum aestivum). International
Journal of Agriculture & Biology 10, 85-88.
Agamy R, Mohamed G, Rady M. 2012. Influence
of the application of fertilizer type on growth, yield,
anatomical structure and some chemical components
of wheat (Triticum aestivum L.) grown in newly
reclaimed soil. Australian Journal of Basic and
Applied Sciences 6, 561-570.
Assuero SG, Tognetti JA. 2010. Tillering
regulation by endogenous and environmental factors
and its agricultural management. The American
Journal of Plant Science and Biotechnology 4, 35-48.
Biswas JC, Ladha JK, Dazzo FB. 2000. Rhizobial
inoculation improves nutritional uptake and growth
of lowland rice. Soil Science Society of America
Journal 64, 1644-1650.
Bric JM, Bostock RM Silverstone SE. 1991. Rapid
in situ assay for indoleacetic acid production by
bacteria immobilized on a nitrocellulose membrane.
Applied and Environmental Microbiology 57, 535-538.
Canbolat MY, Bilen S, Çakmakçı R, Şahin S,
Aydın A. 2006. Effect of plant growth-promoting
bacteria and soil compaction on barley seedling
growth, nutrient uptake, soil properties and
rhizosphere microflora. Biology and Fertility of soils
42, 350-357.
Chen Y, Mei R, Liu L, Kloepper J. 1996. The use
of yield increasing bacteria (YIB) as plant growthpromoting
rhizobacteria in Chinese agriculture.
Management of soil borne diseases.
Ludhiana: Kalyani Publishers 6, 165-184.
Egamberdieva D. 2010.Growth response of wheat
cultivars to bacterial inoculation in calcareous soil.
Plant, Soil and Environment2, 56-58.
Egamberdiyeva D. 2007. The effect of plant growth
promoting bacteria on growth and nutrient uptake of
maize in two different soils. Applied Soil Ecology 36,
184-189.
Gaur A. 1990. Phosphate solubilizing microorganisms
as biofertilizer: Omega Scientific
Publishers.
Hayat R, Ali S, Amara U, Khalid R, Ahmed I.
2010. Soil beneficial bacteria and their role in plant
growth promotion: a review. Annals of microbiology
60, 579-598.
Kang SMO, Khan AL, Hamayun M, Shinwari
ZK, Kim YHA, Joo G, Lee INJ. 2012.
Acinetobacter calcoaceticus ameliorated plant growth
and influenced gibberellins and functional
biochemicals. Pakistan Journal of Botany 44, 365-372.
Khalid A, Arshad M, Zahir Z. 2004. Screening
plant growth‐promoting rhizobacteria for improving
growth and yield of wheat. Journal of Applied
Microbiology 96, 473-480.
Kloepper J, Schroth M, Miller T. 1980. Effects of
rhizosphere colonization by plant growth-promoting
rhizobacteria on potato plant development and yield.
Phytopathology 70, 1078-1082.
Kloepper JW, Zablotowicz RM, Tipping EM,
Lifshitz R. 1991. Plant growth promotion mediated
by bacterial rhizosphere colonizers. The rhizosphere
and plant growth 21, 315-326.
Kohler J, Caravaca F, Carrasco L, Roldán A.
2006. Contribution of Pseudomonas mendocina and
Glomus intraradices to aggregate stabilization and
promotion of biological fertility in rhizosphere soil of
lettuce plants under field conditions. Soil use &
management 22, 298-304.
Mia MAB, Shamsuddin, Z, Wahab Z, Marziah
M. 2010. Effect of plant growth promoting
rhizobacterial (PGPR) inoculation on growth and
nitrogen incorporation of tissue-cultured Musa
plantlets under nitrogen-free hydroponics condition.
Australian Journal of Crop Science 4, 85-90.
37 | Sheirdil et al.

J. Bio. Env. Sci. 2016
Narula N, Deubel A, Gans W, Behl R, Merbach
W. 2006. Paranodules and colonization of wheat
roots by phytohormone producing bacteria in soil.
Plant Soil and Environment 52, 119-121.
Ozturk A, Caglar O, Sahin F. 2003. Yield
response of wheat and barley to inoculation of plant
growth promoting rhizobacteria at various levels of
nitrogen fertilization. J. of Plant Nutrition & Soil
Science 166, 262-266.
Pikovskaya R. 1948. Mobilization of phosphorus in
soil in connection with vital activity of some microbial
species. Mikrobiologiya 17, 362-370.
Rodríguez H, Fraga R. 1999. Phosphate
solubilizing bacteria and their role in plant growth
promotion. Biotechnology advances 17, 319-339.
Rolfe BG, Dazzo FB, Biswas JC, Ladha JK,
Yann YG. 2000. Rhizobial inoculation influences
seedling vigor and yield of rice. Agronomy Journal
92, 880-886.
Saber Z, Pirdashti H, Esmaeili M, Abbasian A,
Heidarzadeh A. 2012. Response of Wheat Growth
Parameters to Coinoculation of Plant Growth
Promoting Rhizobacteria (PGPR) and Different
Levels of Inorganic Nitrogen and Phosphorus. World
Applied Sciences Journal 16, 213-219.
Sadaghiani MHR, Barin M, Jalili F. 2008. The
Effect of PGPR inoculation on the growth of wheat.
International Meeting on Soil Fertility Land
Management and Agroclimatology. Turkey, 891-898
Steel RGD, Torrie JH, Dickey DA. 1980.
Principles and procedures of statistics: a biometrical
approach. Mc Graw-Hill, Inc 113, 137-154.
Turan M, Gulluce M, Cakmakci R, Oztas T
Sahin F. 2010. The effect of PGPR strain on wheat
yield and quality parameters. 19 th World Congress of
Soil Science, Soil Solutions for a Changing World: 1-6
August 2010, Brisbane, Australia.
Upadhyay SK, Singh JS, Saxena AK, Singh DP.
2011. Impact of PGPR inoculation on growth and
antioxidant status of wheat under saline conditions.
Plant Biology 12, 256-258.
Van Loon L. 2007. Plant responses to plant growthpromoting
rhizobacteria. European Journal of Plant
Pathology 119, 243-254.
Vessey JK. 2003. Plant growth promoting
rhizobacteria as biofertilizers. Plant & soil 255, 571-586.
Watanabe F, Olsen S. 1965. Test of an Ascorbic
Acid Method for Determining Phosphorus in Water
and NaHCO3 Extracts from Soil 1. Soil Science 29,
677-678.
Wu S, Jia S, Sun D, Chen M, Chen X, Zhong J,
Huan L. 2005. Purification and characterization of
two novel antimicrobial peptides Subpeptin JM4-A
and Subpeptin JM4-B produced by Bacillus subtilis
JM4. Current microbiology 51, 292-296.
Zhang J, Liu J, Meng L, Ma Z, Tang X, Cao Y,
Sun L. 2012. Isolation and characterization of plant
growth-promoting rhizobacteria from wheat roots by
wheat germ agglutinin labeled with fluorescein
isothiocyanate. The Journal of Microbiology 50, 191-
198.
38 | Sheirdil et al.